Solid pharmaceutical compositions comprising biopterin derivatives and uses of such compositions

ABSTRACT

The present invention relates to a method of preparing 4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride dihydrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/584,890, filed May 2, 2017, which is a continuation of U.S.patent application Ser. No. 14/913,665, now granted U.S. Pat. No.9,895,372, issued Feb. 20, 2018, which was filed under 35 U.S.C. § 371as the U.S. national phase of International Application No.PCT/EP2015/056824, filed Mar. 30, 2015, which designated the U.S. andclaims the benefit of priority to European Patent Application No.14162727.3, filed Mar. 31, 2014, each of which is hereby incorporated inits entirety including all tables, figures and claims.

FIELD OF THE INVENTION

The present invention relates to solid pharmaceutical compositionscomprising biopterin derivatives as well as methods for obtaining suchsolid pharmaceutical compositions. The invention also relates to thesolid pharmaceutical compositions of the invention for treatingdiseases.

BACKGROUND OF THE INVENTION

Biopterines and their derivatives are molecules of pharmaceuticalinterest. For example, tetrahydrobiopterin (BH4, sapropterin) hasrecently been approved for treatment of hyperphenylalaninaemia (HPA) inadult and paediatric patients of 4 years of age and over withphenylketonuria (PKU). For this purpose, tetrahydrobiopterin isformulated in the commercial drug (sold under the name Kuvan) as adissolvable tablet with Mannitol (E421), Calcium hydrogen phosphate,anhydrous, Crospovidone type A, Ascorbic acid (E300), Sodium stearylfumarate and Riboflavin (E101). See in this context also WO 2006/055511.

Other therapeutically promising biopterin compounds are4-Amino-5,6,7,8-tetrahydro-L-biopterin and4-Amino-7,8-dihydro-L-biopterin. Both compounds have been shown toexhibit properties different from that of other NO-inhibitors, makingthe compound potentially more suitable than “classical” arginineanalogues (Werner et al., (1996). Biochemical Journal 320, 93-6 or U.S.Pat. No. 5,922,713). Both compounds have been shown to be effective inexperimental TBI (see, for example, WO 2004/084906, U.S. Pat. No.8,222,828, European Patent 0 906 913 or Terpolilli et al., JNeurotrauma. 2009; 26 (11):1963-75). However, these biopterinderivatives have not yet been approved for medical treatment. Thus,there is a need to provide pharmaceutical compositions that are suitablefor therapeutic applications in humans. Ideally, such a pharmaceuticalcomposition should be easy to prepare, easy to use and yet stable—it isnoted in this context that hydrogenated biopterin derivatives aresensitive against oxidization, when stored over a long time or whenprovided in solution.

It is thus an object of the present invention to provide apharmaceutical composition containing biopterin derivatives that fulfilsthese needs.

SUMMARY OF THE INVENTION

This problem is solved by the embodiments of the invention as defined inthe claims, described in the description, and illustrated in theExamples and Figures.

The present invention relates in one embodiment to a solidpharmaceutical composition (adapted for intravenous administration)comprising

-   a) a compound having the formula (I):

-   -   and/or a compound having the formula (II):

-   and-   b) at least one phosphate salt.

The solid composition of the present invention may be “adapted forintravenous administration”. This means that, after the solidcomposition is mixed with a pharmaceutically acceptable carrier,preferably a pharmaceutically acceptable fluid, e.g. water or buffer orany reconstitution fluid, a composition is obtained which directly assuch can be used for intravenous application. Thus, after the mixing ofthe solid composition with the pharmaceutically acceptable carrier aready-to-use composition, which is suitable for intravenous application,is obtained.

In one embodiment in the pharmaceutical composition of the presentinvention, the at least one phosphate salt is a sodium phosphate, apotassium phosphate or an ammonium phosphate. The phosphate salt may beselected from the group consisting of Na₂HPO₄ (water free),Na₂HPO₄.2H₂O, Na₂HPO₄.7H₂O, Na₂HPO₄.12H₂O, NaH₂PO₄ (water free),NaH₂PO₄.H₂O, NaH₂PO₄.2H₂O, K₂HPO₄ (water free) K₂HPO₄.3H₂O, KH₂PO₄(water free) and mixtures thereof.

In some embodiments of the pharmaceutical composition of the presentinvention, the phosphate salt is Na₂HPO₄.2H₂O and the quantity of theNa₂HPO₄.2H₂O present in the composition is chosen such that the molarratio of the Na₂HPO₄.2H₂O to compound (I) or compound (II) ranges from0.04 to 0.4.

In further embodiments of the pharmaceutical composition of the presentinvention, the sodium phosphate is NaH₂PO₄.2H₂O, and the quantity of theNaH₂PO₄.2H₂O present in the composition is chosen such that the molarratio of the NaH₂PO₄.2H₂O to compound (I) or compound (II) ranges from0.01 to 0.09.

In other embodiments, the pharmaceutical composition of the presentinvention comprises two different sodium phosphate salts. Optionally,the two different sodium phosphate salts are NaH₂PO₄.2H₂O andNa₂HPO₄.2H₂O.

In some embodiments of the pharmaceutical composition of the presentinvention, the quantity of the NaH₂PO₄.2H₂O and Na₂HPO₄.2H₂O present inthe composition is chosen such that the molar ratio of both NaH₂PO₄.2H₂Oand Na₂HPO₄.2H₂O to compound (I) or compound (II) ranges from 0.02 to0.5.

In some embodiments of the pharmaceutical composition of the presentinvention, the quantity of the NaH₂PO₄.2H₂O and Na₂HPO₄.2H₂O present inthe composition is chosen such that the molar ratio of each ofNaH₂PO₄.2H₂O and Na₂HPO₄.2H₂O to compound (I) or compound (II) rangesfrom 0.02 to 0.5.

In further embodiments of the pharmaceutical composition of the presentinvention the compound (I) and/or the compound (II) are present as thefree base.

In some embodiments of the pharmaceutical composition of the presentinvention the pharmaceutical composition is a lyophilized pharmaceuticalcomposition.

In another embodiment of the pharmaceutical composition of the presentinvention, the compound (I) is a compound having the formula (Ia):

In another embodiment of the pharmaceutical composition of the presentinvention, the compound (II) is a compound having the formula (IIa):

In some embodiments of the pharmaceutical composition of the presentinvention, the pharmaceutical composition comprises an additionalpharmaceutical excipient.

In another embodiment of the pharmaceutical composition of the presentinvention, the additional pharmaceutical excipient is an inorganic salt.The inorganic salt can be selected from MgCl₂, CaCl₂, NH₄Cl, KCl, orNaCl, preferably the inorganic salt is NaCl.

In some embodiments of the pharmaceutical composition of the presentinvention, the quantity of the NaCl present in the composition of thepresent invention is chosen such that the molar ratio of the NaCl tocompound (I) or compound (II) ranges from 1.5 to 4, preferably from 1.8to 3.7.

In some embodiments the pharmaceutical composition of the presentinvention further comprises crystallization water.

In other embodiments the pharmaceutical composition of the presentinvention is adapted to be reconstituted in water. “Adapted to bereconstituted in water” means that (a dehydrated or concentrated)composition can be returned to the liquid state by adding water.

In further embodiments the pharmaceutical composition of the presentinvention is adapted for administration by infusion or injection.

In some embodiments of the pharmaceutical composition of the presentinvention the compound (I) is(6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin.

In further embodiments of the pharmaceutical composition of the presentinvention the compound (I) is(6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin.

In other embodiments of the pharmaceutical composition of the presentinvention the compound (I) is a diastereomeric mixture that comprisesmore (6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin than(6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin.

In some embodiments of the pharmaceutical composition of the presentinvention the quantity of the(6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin and the(6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin is chosen such that theratio of the amount of (6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin tothe (6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin ranges from 0.5 to 2,preferably around 1.3.

In further embodiments of the pharmaceutical composition of the presentinvention, a unit dosage of the composition contains 650±60 mg of thefree base of 4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin, 140±30 mg ofwater of crystallization, 70±7 mg disodium hydrogen phosphate dihydrate(Na₂HPO₄.2H₂O), 16.5±2 mg sodium dihydrogen phosphate dihydrate(NaH₂PO₄.2H₂O), and 350±30 mg sodium chloride (NaCl).

In other embodiments of the pharmaceutical composition of the presentinvention, a unit dosage of the composition contains 650±60 mg of thefree base of 4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin, 60±50 mg ofwater of crystallization, 70±7 mg disodium hydrogen phosphate dihydrate(Na₂HPO₄.2H₂O), 12±2.5 mg sodium dihydrogen phosphate dihydrate(NaH₂PO₄.2H₂O), and 350±30 mg sodium chloride (NaCl).

In some embodiments of the pharmaceutical composition of the presentinvention the composition comprises 1, 2, 3, 4, 5, 6, 7, or moreadditional compounds, wherein the additional compounds are selected fromthe group consisting of one or more of the compounds selected from thegroup consisting of 4-Amino-L-biopterin,(6R,S)-5,6,7,8-Tetrahydro-L-biopterin,1-[(6R,S)-2,4-Diamino-5,6,7,8-tetrahydropteridin-6-yl]propanol,1-[(6R,S)-2,4-Diamino-5,6,7,8-tetrahydropteridin-6-yl]propane,(1R,2S)-1-[(6R,S)-2-(Acetylamino)-4-amino-5,6,7,8-tetrahydropterin-6-yl]-1,2-diacetoxy-propane,2,4-Diamino-7,8-dihydropteridine, 2,4-Diaminopteridine.

The present invention also relates to a use of a lyophilizedpharmaceutical composition of the present invention for the treatment ofa disease. In some embodiments, the disease is selected from the groupconsisting of a traumatic brain injury, non-traumatic brain injury,preferably stroke or meningitis, elevated cranial pressure, secondarybrain injury.

Also, the present invention relates to a lyophilized pharmaceuticalcomposition for use in the treatment of a disease. In some embodiments,the disease is selected from the group consisting of a traumatic braininjury, non-traumatic brain injury, preferably stroke or meningitis,elevated cranial pressure, secondary brain injury.

The present invention further relates to a method for preparing alyophilized solid pharmaceutical composition (adapted for intravenousadministration) comprising

-   a) a compound having the formula (I):

-   -   and/or a compound having the formula (II):

-   and-   b) at least one phosphate salt and optionally NaCl;-   the method comprising:-   aa) dissolving a compound of the formula (III) and/or the compound    of the formula (II):

-   -   with a buffer, wherein preferably the buffer comprises the        phosphate;

-   bb) lyophilization of the solution obtained in aa).

In some embodiments, the method of the present invention furthercomprises the step of dissolving the lyophilisate obtained in bb) in apharmaceutically acceptable fluid for the preparation of an injectablesolution.

Accordingly, the present invention also relates to a method forpreparing an injectable solution comprising:

-   a) a compound having the formula (I):

-   -   and/or a compound having the formula (II):

-   and-   b) at least one phosphate salt and optionally NaCl;-   the method comprising:-   aa) dissolving the compound of the formula (III) and/or (II):

-   -   with a buffer, wherein preferably the buffer comprises the        phosphate;

-   bb) lyophilization of the solution obtained in aa);

-   cc) reconstituting the lyophilisate obtained in bb) in a    pharmaceutically acceptable fluid for the preparation of an    injectable solution, wherein the lyophilisate obtained in bb) is    filled into a vial.

In one embodiment of the method of the present invention thelyophilisate obtained in bb) is filled into a 50 ml vial,

-   (i) preferably in an amount about 1-1.5 g, preferably 1.25 g solid    formulation; or-   (ii) preferably in an amount about 0.9-1.4 g, preferably 1.15 g    solid formulation.

In other embodiments in the method of the present invention the bufferin aa) is a sodium hydrogen phosphate buffer comprising at least onephosphate salt.

In other embodiments in the method of the present invention the bufferin aa) comprises NaOH, sodium hydrogen phosphate buffer and water.Optionally, the NaOH is a 5 N NaOH dissolution.

In another embodiment of the method of the present invention the sodiumhydrogen phosphate buffer is prepared by separately dissolvingNaH₂PO₄.2H₂O and Na₂HPO₄.2H₂O.

In a further embodiment of the method of the present invention thesodium hydrogen phosphate buffer has a pH of 7.4 by adding theNaH₂PO₄.2H₂O dissolution to the Na₂HPO₄.2H₂O dissolution.

In some embodiments of the method of the present invention the buffercomprises 12-16% (w/w) NaOH 5N, 8-12% (w/w) sodium hydrogen phosphatebuffer and 74-78% (w/w) water for injection.

In another embodiment of the method of the present invention, thesolution obtained in aa) is sterile filtered, preferably with a 0.22 μmfilter.

In further embodiments of the method of the present invention, thebuffer has a pH of about 8, 9, 10, 11, 12, 13 or 14.

In yet another embodiment of the method of the present invention, thesolution in step aa) has a pH of about 4, 5, 6, 7, 8, 9, 10 or 11preferably between 6.5-7.6, most preferably 7.4.

In other embodiments of the method of the present invention, thelyophilisate obtained in bb) is filled into vials, preferably in anamount about 1-1.5 g, preferably 1.25 g solid formulation; or preferablyin an amount about 0.9-1.4 g, preferably 1.15 g solid formulation.

In further embodiments of the method of the present invention the bufferis prepared with degassed buffer. In some embodiments, the buffer isdegassed with nitrogen until the oxygen content is <1.0 ppm.

In another embodiment of the method of the present invention after thepreparation of the solution; the lyophilisation is started at most 2hours later.

The present invention also relates to a pharmaceutical compositionobtainable by the method of the present invention.

The present invention further relates to the use of the lyophilizedpharmaceutical composition of the present invention in the manufactureof a medicament for treating a subject having traumatic brain injurysuch as closed head injury, elevated cranial pressure but also secondarybrain injury or non-traumatic brain injury such as stroke or meningitis.

Also, the present invention relates to a method of treating a disease ina subject, comprising the step of administering a lyophilizedpharmaceutical composition of the present invention to a subject in needthereof. In some embodiments, in the method of treating a disease in asubject the maximal daily dose is 20 mg/kg body weight and day,preferably, 17.5, 15.0, or 12.5, 10, 8.5, 7.5, 5.0, or 2.5 mg/kg bodyweight and day.

The present invention also relates to a lyophilized pharmaceuticalcomposition for use in treating a disease in a subject, the usecomprising the step of administering a lyophilized pharmaceuticalcomposition of the present invention to a subject in need thereof. Insome embodiments, the maximal daily dose is 20 mg/kg body weight andday, preferably, 17.5, 15.0, or 12.5, 10, 8.5, 7.5, 5.0, or 2.5 mg/kgbody weight and day.

In yet another embodiment the invention relates to a solidpharmaceutical composition (adapted for intravenous administration)comprising

-   a) a compound having the formula (I):

-   -   and/or a compound having the formula (II):

-   and-   b) at least one inorganic salt, preferably NaCl.

In such an embodiment, the quantity of the NaCl present in thecomposition of the present invention may be chosen such that the molarratio of the NaCl to compound (I) or compound (II) ranges from 1.5 to 4,preferably from 1.8 to 3.7, from 1.85 to 3.6, from 1.9 to 3.4, mostpreferably from 1.9 to 2.5.

In one embodiment the molar ratio of NaCl to compound (I) or compound(II) is about 2.2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the preparation of4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride dihydrate(VAS203) drug substance.

FIG. 2 shows a manufacturing process of VAS203 drug product according tothe invention.

FIG. 3 shows the stability of 1 g VAS203 vials (batch no. 928606),stored at 2-8° C. (Table 5).

DETAILED DESCRIPTION OF THE INVENTION

It was an object of the present invention to provide pharmaceuticalcompositions. Additionally, these compositions should be easy to use andstable. It has been found herein that by using solid phosphate salts(alone or in connection with an inorganic salt such as NaCl or KCl) inconnection with solid biopterin derivatives such as the compounds offormula (I) or (II) results in pharmaceutical compositions(formulations) that are very well adapted to achieve a pH value or pHrange as well as an osmolality that, upon dissolution, is directlysuitable for therapeutic administration. The solid pharmaceuticalcompositions are particularly well suited and easy to use, since theyare already adapted for intravenous administration, thereby alsoensuring save usage. In addition, the problem of oxidation of biopterinderivatives in liquids is being solved by the provision of solidcompositions.

Thus, the present invention relates to a solid pharmaceuticalcomposition (adapted for intravenous administration) comprising

-   a) a compound having the formula (I):

-   -   and/or a compound having the formula (II):

-   and-   b) at least one phosphate salt.

In addition, the present invention also relates to a solidpharmaceutical composition (adapted for intravenous administration)comprising

-   a) a compound having the formula (I):

-   -   and/or a compound having the formula (II):

-   and-   b) at least one inorganic salt, in particular NaCl.

The term “solid” or “solid composition” when used herein relates toparticles (ions, atoms or molecules or compounds) which are closelypacked together. The forces between particles are strong so that theparticles cannot move freely but can only vibrate. As a result, a solidhas a stable, definite shape, and a definite volume. Solids can onlychange their shape by force, as when broken or cut. In crystallinesolids, the particles (atoms, molecules, or ions) are packed in aregularly ordered, repeating pattern. There are various differentcrystal structures, and the same substance can have more than onestructure (or solid phase). The term “solid” also encompasses amorphousor non-crystalline compositions/substances/solids. Usually, theaggregate state is determined at room temperature and ambient pressure.Room temperature commonly represents the small range of temperatures atwhich the air feels neither hot nor cold, often denoted is the rangebetween 20 and 23.5° C. with an average of 21° C. (70° F.). With ambientpressure is meant a pressure between 900 and 1200 hPa (hekto Pascal),preferably about 1000 hPa. Solids can be transformed into liquids bymelting, and liquids can be transformed into solids by freezing. Solidscan also change directly into gases through the process of sublimation.

The solid composition of the present invention can be administered(usually upon dissolution in a liquid such as water) to an individual(“administration”). This provides administration of a therapeuticallyeffective dose of the solid composition of the present invention to asubject.

The “therapeutically effective amount” is a dose of the compound offormula (I) and/or the compound of formula (II) that produces theeffects for which it is administered. The exact dose will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques. As is known in the art and describedabove, adjustments for systemic versus localized delivery, age, bodyweight, general health, sex, diet, time of administration, druginteraction and the severity of the condition may be necessary, and willbe ascertainable with routine experimentation by those skilled in theart. In this context, it is noted again that both compounds,4-Amino-5,6,7,8-tetrahydro-L-biopterin and4-Amino-7,8-dihydro-L-biopterin are pharmaceutically active. It is alsonoted that 4-Amino-7,8-dihydro-L-biopterin can be obtained by oxidation(also spontaneous oxidation) from4-Amino-5,6,7,8-tetrahydro-L-biopterin. Thus, a composition of theinvention can comprise either only4-Amino-5,6,7,8-tetrahydro-L-biopterin or4-Amino-7,8-dihydro-L-biopterin or mixture of these two compounds in anyratio.

The solid compositions of the present invention are applicable to bothhuman therapy and veterinary applications. The compounds describedherein having the desired therapeutic activity may be administered in apharmaceutically acceptable carrier to a patient/subject, as describedherein. Depending upon the manner of introduction, the compounds may beformulated in a variety of ways as discussed below. The solidcomposition of the present invention maybe administered alone or incombination with other treatments.

The solid composition of the present invention may be further “adaptedfor intravenous administration”. This means that, after the solidcomposition is preferably mixed with a pharmaceutically acceptablecarrier, preferably a pharmaceutically acceptable fluid, e.g. water orbuffer or any of the reconstitution fluid as described below, acomposition is obtained which directly as such can be used forintravenous application. Thus, after the mixing of the solid compositionwith the pharmaceutically acceptable carrier a ready-to-use composition,which is suitable for intravenous application, is obtained. Intravenousadministration is the infusion or injection of liquid substancesdirectly into a vein usually with a syringe and a hollow needle which ispierced through the skin to a sufficient depth for the material to beadministered into the body of the subject.

Thus, in further embodiments the pharmaceutical composition of thepresent invention is adapted for administration by infusion orinjection. With an “infusion” is meant a continuous administration overa certain period of time. For example such an administration may take inbetween 10 minutes to 4 days. Thus it can take at most 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 32,40, 48, 56, 68, 72, 86, or 96 hours. An “injection” means is a transientinfusion method of putting fluid into the body of a subject. Usuallysuch an administration takes less than 10 minutes. However, an injectioncan be repeated various times a day. For example, an injection may takeplace 1, 2, 3, 4, 5, 6, 7, 8 or 9 times a day. Further the injection(s)may be administered for 1, 2, 3 or 4 days. However, the administrationvia injection or infusion can also take longer if needed. It is clearthat the exact duration depends on many factors.

Typically, compositions for intravenous administration are the solidpharmaceutical composition of the present invention mixed with apharmaceutically acceptable carrier, for example, with sterile isotonicaqueous buffer to form a pharmaceutical solution. Where necessary, thiscomposition/solution may also include a solubilizing agent and a localanaesthetic such as lignocaine to ease pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a dry lyophilisedpowder or water free concentrate in a hermetically sealed container suchas a vial, an ampoule or sachette indicating the quantity of compound(I) and/or compound (II). Where the composition/solution is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection or infusion, an ampoule ofsterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

The term “pharmaceutically acceptable” means approved by a regulatoryagency or other generally recognized pharmacopoeia for use in animals,and more particularly in humans. Generally “pharmaceutically acceptable”means also “physiologically acceptable” which means that thefluid/carrier is in accord with or characteristic of the normalfunctioning of a living subject and does not provoke toxicity or anyother adverse effects. Such a fluid/carrier normally has about the samepH and/or osmolality as e.g. fluids such as the blood of an animal.

The “pharmaceutically acceptable fluid” can be one of the followingnon-limiting list of non-aqueous or aqueous solvents. Non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Aqueous solvents include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media, sodium ionsolution, Ringer's dextrose, dextrose and sodium ion, lactated Ringer's,or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. A preferred pharmaceutically acceptable fluidis an aqueous solvent for example sterile water for injection.

In some embodiments, the solid pharmaceutical composition of the presentinvention is reconstituted before administration. In other embodiments,the pharmaceutical composition of the present invention is adapted to bereconstituted in water. “Reconstituted in water” means to return (adehydrated or concentrated) composition to the liquid state by adding apharmaceutically acceptable fluid as described above. Preferably, thesolid composition of the present invention is reconstituted in water.\

The invention may comprise different solid compositions. For example,the solid composition can comprise compound (I) and/or compound (II) inaddition to a phosphate salt. By a “phosphate salt” is meant that anyknown phosphate salt can be used. Phosphate salts refer to manydifferent combinations of the chemical phosphate with salts andminerals. In one embodiment in the pharmaceutical composition of thepresent invention, the at least one phosphate salt is a sodiumphosphate, a potassium phosphate or an ammonium phosphate. The phosphatesalt can be selected from the group consisting of Na₂HPO₄ (water free),Na₂HPO₄.2H₂O, Na₂HPO₄.7H₂O, Na₂HPO₄.12H₂O, NaH₂PO₄ (water free),NaH₂PO₄.H₂O, NaH₂PO₄.2H₂O, K₂HPO₄ (water free) K₂HPO₄.3H₂O, KH₂PO₄(water free) and mixtures thereof.

In some embodiments of the pharmaceutical composition of the presentinvention, the phosphate salt is Na₂HPO₄.2H₂O and the quantity of theNa₂HPO₄.2H₂O present in the composition is chosen such that the molarratio of the Na₂HPO₄.2H₂O to compound (I) or compound (II) ranges from0.04 to 0.4, preferably from 0.05 to 0.35, from 0.075 to 0.25, from 0.09to 0.2. In one embodiment the molar ratio of the Na₂HPO₄.2H₂O tocompound (I) or compound (II) is about 0.144.

In further embodiments of the pharmaceutical composition of the presentinvention, the sodium phosphate is NaH₂PO₄.2H₂O, and the quantity of theNaH₂PO₄.2H₂O present in the composition is chosen such that the molarratio of the NaH₂PO₄.2H₂O to compound (I) or compound (II) ranges from0.01 to 0.09 preferably from 0.015 to 0.07, from 0.02 to 0.05, from0.025 to 0.04, from 0.035 to 0.05. In one embodiment the molar ratio ofthe NaH₂PO₄.2H₂O to compound (I) or compound (II) is about 0.038.

In other embodiments, the pharmaceutical composition of the presentinvention comprises two different sodium phosphate salts. Optionally,the two different sodium phosphate salts are NaH₂PO₄.2H₂O andNa₂HPO₄.2H₂O.

In some embodiments of the pharmaceutical composition of the presentinvention, the quantity of the NaH₂PO₄.2H₂O and Na₂HPO₄.2H₂O present inthe composition is chosen such that the molar ratio of both NaH₂PO₄.2H₂Oand Na₂HPO₄.2H₂O to compound (I) or compound (II) ranges from 0.02 to0.5, preferably from 0.03 to 0.45, from 0.04 to 0.3, from 0.05 to 0.25,from 0.06 to 0.2, from 0.07 to 0.15. In one embodiment the molar ratioof both NaH₂PO₄.2H₂O and Na₂HPO₄.2H₂O to compound (I) or compound (II)is about 0.18.

In some embodiments of the pharmaceutical composition of the presentinvention, the quantity of the NaH₂PO₄.2H₂O and Na₂HPO₄.2H₂O present inthe composition is chosen such that the molar ratio of each ofNaH₂PO₄.2H₂O and Na₂HPO₄.2H₂O to compound (I) or compound (II) rangesfrom 0.02 to 0.5, preferably from 0.025 to 0.4, from 0.025 to 0.3, from0.025 to 0.2, from 0.03 to 0.1. In some embodiments the quantity of theNaH₂PO₄.2H₂O present in the composition is chosen such that the molarratio of the NaH₂PO₄.2H₂O to compound (I) or compound (II) ranges from0.01 to 0.09 and the quantity of the Na₂HPO₄.2H₂O present in thecomposition is chosen such that the molar ratio of the Na₂HPO₄.2H₂O tocompound (I) or compound (II) ranges from 0.04 to 0.4. Here, also theother quantities of the singly applied phosphate salts Na₂HPO₄.2H₂O andNaH₂PO₄.2H₂O as described above apply. In some embodiments, the solidpharmaceutical composition comprises least 1, 2, 3, 4, 5, 6, or morephosphate salts. In one embodiment the solid pharmaceutical compositionof the present invention comprises 2 (different) phosphate salts.

When used herein, the term “about” is understood to mean that there canbe variation in the respective value or range (such as pH,concentration, percentage, molarity, time etc.) that can be up to 5%, upto 10%, up to 15% or up to and including 20% of the given value. Forexample, if a formulation comprises about 5 mg/ml of a compound, this isunderstood to mean that a formulation can have between 4 and 6 mg/ml,preferably between 4.25 and 5.75 mg/ml, more preferably between 4.5 and5.5 mg/ml and even more preferably between 4.75 and 5.25 mg/ml, with themost preferred being 5 mg/ml. The same applies also to molarity. Forexample a molarity of about 1 is to be understood as a molarity ofbetween 0.8 to 1.2, preferably 0.85 to 1.15, more preferably between 0.9to 1.1, even more preferably between 0.95 to 1.05 with the mostpreferred being 1.

Alternatively, the present invention relates to solid compositions,which comprise compound (I) and/or compound (II) and an inorganic saltdifferent from a phosphate salt. In these compositions, the inorganicsalt can either be the sole components apart from compound (I) or (II)(in this case, there is no phosphate salt present). Alternatively, incomposition of the invention an inorganic salt can be present incombination with at least one phosphate. The term “inorganic salt” whenreferred to herein means every suitable inorganic salt. Optionally, theinorganic salt is selected from MgCl₂, CaCl₂, NH₄Cl, KCl, or NaCl. Insome preferred embodiments the inorganic salt is NaCl. In someembodiments of the pharmaceutical composition of the present inventionthe quantity of the NaCl present in the composition of the presentinvention (alone or together with at least one phosphate) is chosen suchthat the molar ratio of the NaCl to compound (I) or compound (II) rangesfrom 1.5 to 4, preferably from 1.8 to 3.7, from 1.85 to 3.6, from 1.9 to3.4, most preferably from 1.9 to 2.5. In one of such embodiments themolar ratio of the NaCl to compound (I) or compound (II) is about 2.2

In the compositions of the invention, compounds (I) and/or compound (II)can be present as diastereomeric mixtures or mixtures of 1, 2, 3, 4, 5,6, 7 or 8 stereoisomers for compound (I) and 1, 2, 3, 4, 5 or 6stereoisomers for compound (II), preferably of one or two stereoisomers.

The compound of formula (I) can thus comprise diastereomeric mixtures ofcompound of formula (I) or mixtures of one or more stereoisomers of thecompound of formula (I). The compound (I) can be(6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin. In further embodiments thecompound (I) is (6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin. In otherembodiments the compound (I) is a diastereomeric mixture that comprisesmore (6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin than(6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin. In some embodiments thequantity of the (6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin and the(6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin is chosen such that theratio of the amount of (6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin tothe (6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin ranges from 0.5 to 2,preferably from 0.5 to 1.9, from 0.7 to 1.8, from 0.8 to 1.7, from 0.9to 1.6, from 1 to 1.5, most preferably from 1.1 to 1.4. In oneembodiment, the quantity of the(6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin and the(6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin is chosen such that theratio of the amount of (6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin tothe (6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin is around 1.3. In someembodiments, the pharmaceutical composition of the present inventioncomprises only compound (I).

The compound (I) can also be a compound having the formula (Ia):

Similarly, the compound of formula (II) when referred to herein cancomprise stereoisomeric mixtures of compound of formula (II) or mixturesof one or more stereoisomer of the compound of formula (II). In someembodiments of the pharmaceutical composition of the present inventionthe compound (II) is 4-Amino-7,8-dihydro-L-biopterin. In anotherembodiment, the compound (II) is a compound having the formula (IIa):

In some embodiments, the pharmaceutical composition of the presentinvention comprises only compound (II).

Within the solid pharmaceutical compositions compound (I) and/or thecompound (II) can also be present as the free base. A “free base” whenreferred to herein refers to the pure basic form of an amine, as opposedto its salt form. Further, this term is used to describe thedeprotonated amine form of a compound. A common counter ion is an ionfrom an inorganic acid such as a negatively charged chloride. Forexample, compare the free base amine (NH₂) with amine hydrochloride (NH₃⁺ Cl⁻) when adding HCl.

As mentioned above, the biopterin derivatives are sensitive againstoxidation in liquids. Therefore, the solid pharmaceutical compositioncan also be provided as a lyophilized pharmaceutical composition. Theterm “lyophilized” when used herein means freeze-drying, which is adehydration process. Freeze-drying works by freezing the material andthen reducing the surrounding pressure to allow the frozen water in thematerial to sublimate directly from the solid phase to the gas phase. Insome embodiments the final residual water content in the lyophilizedproduct is between 0% to 15%, preferably between 0% to 12%, (w/w).Methods to perform lyophilisation are known to the person skilled in theart.

As evident for the person skilled in the art, the pharmaceuticalcompositions can further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreadditional pharmaceutical excipients. A “pharmaceutical excipient” oradditives are compounds added to the compounds of the formula (I) and/or(II). These additives might serve a specific function. They may be addedto increase bulk, aid manufacturing, improve stability, enhance drugdelivery and targeting, and modify drug safety or pharmacokineticprofile. Ingredients that are used during drug product manufacturing butmay not be present in the solid composition of the present invention arealso considered excipients (examples include water for lyophilizedproducts, and inert gases in the head space of containers). Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,trehalose, mannitol, sorbitol, glycine, histidine, raffinose, gelatine,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium ion, dried skim milk, glycerol, propylene,glycol, dextrose, dextran, water, ethanol and the like. The composition,if desired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion and the like.

If, for example, the solid composition of the present inventioncomprises a compound of formula (I) and/or of formula (II) and aphosphate salt, a possible additional pharmaceutical excipient can be aninorganic salt. The inorganic salt can be any inorganic salt asdescribed above. On the other hand, if the pharmaceutical composition ofthe present invention, comprises a compound of formula (I) and/or offormula (II) and a inorganic salt, an additional pharmaceuticalexcipient can be a phosphate salt. The phosphate salt can be anyphosphate salt as described above.

In addition to the pharmaceutical excipients which may be present in thesolid pharmaceutical compositions of the present inventions, in someembodiments the pharmaceutical composition of the present invention cancomprise crystallization water, of course an additional pharmaceuticalexcipients can also be present as well. As used herein in accordancewith its regular meaning in the art, “water of crystallization” or“water of hydration” or “crystallization water” means water that occursinside crystals—in the present invention, both the inorganic salts suchas Na₂HPO₄ or NaH₂PO₄ as well as the biopterin derivate of compound (I)or compound (II) can have water of crystallization. While in the solidphosphate salts such as Na₂HPO₄.2H₂O, or NaH₂PO₄.2H₂O the water ofcrystallization is present in defined stoichiometric amounts, the amountof water of crystallization present in the solid form of compound (I) orcompound (II) may vary depending on the conditions for the synthesisand/or crystallization of the compound that is used for the preparationof the solid formulation of the present invention. Without wishing to bebound by theory, it is believed that the water of crystallization thatis present in the solid form of compound (I) or compound (II) can, forexample, be bound via hydrogen bonds to the two amino groups of compound(I) or (II) which are present as free base. For illustrative purposes,it referred in this context to a unit dosage of the composition thatcontains 650±60 mg of the free base of4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin, 140±30 mg of water ofcrystallization, 70±7 mg Na₂HPO₄.2H₂O, 16.5±2 mg NaH₂PO₄.2H₂O, and350±30 mg sodium chloride (NaCl). In this unit dosage the two sodiumphosphate salts are present as dihydrates, while the crystallizationwater that is present in the amount of 140±30 mg of water ofcrystallization only refers to the water that present in associationwith the free base of compound (I) and/or compound (II). Anotherexemplary unit dosage of the composition contains 650±60 mg of the freebase of 4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin, 60±50 mg of waterof crystallization, 70±7 mg Na₂HPO₄.2H₂O, 12±2.5 mg NaH₂PO₄.2H₂O, and350±30 mg sodium chloride (NaCl).

As just described, the pharmaceutical composition of the presentinvention may be provided in a unit dosage. A “unit dosage” of the solidpharmaceutical compositions of the present invention means that theingredients are mixed together in a unit dosage, typically involving amixture of active drug components such as compound (I) and/or compound(II) and nondrug components (excipients) such as at least one phosphatesalt and/or at least one inorganic salt. Additionally, a unit dosage maycomprise further excipients such as at least one phosphate salt or atleast one inorganic salt and/or along other non-reusable material thatmay not be considered either ingredient or packaging (such as a capsuleshell, for example). The term unit dosage can also encompassnon-reusable packaging as well (especially when each drug product isindividually packaged). The unit dosage can also comprise reconstitutedsolid pharmaceutical compositions of the present inventions.

For an exemplary unit dosage in the following a calculation of the molarratios of the ingredients in this unit dosage is outlined in Table 1.

TABLE 1 Composition Molar mass Mol (mean) form C₉H₁₆N₆O₂ Compound of 650+/− 60 mg 240.27 g/mol 0._0027053 solid formula (i) C₉H₁₄N₆O₂ Compoundof 650 +/− 60 mg 238.26 g/mol 0.0027282 solid formula (ii) H₂O Water of140 +/− 30 mg  18.02 g/mol 0.0077691 crystallization NaCl Sodium 350 +/−30 mg  58.44 g mol−1 0.005989 salt chloride Na₂HPO₄•2H₂O Disodium  70+/− 7 mg 177.99 g/mol 0.0003932 Dihydrate hydrogen solid phosphateDihydrate NaH₂PO₄•2H₂O Sodium  16.5 +/− 2 mg 156.01 g/mol 0.0001057Dihydrate dihydrogen solid phosphate Dihydrate

Molar ratio between n_(NACl)/n_(compound (i))=0.005989 mol/0.0027053mol˜2.214

Molar ratio between n_(NACl)/n_(compound (ii))=0.005989 mol/0.0027282mol˜2.195

Molar ratio betweenn_(Na2HPO4.2H2O)+n_(NaH2PO4.2H2O/)n_(compound (i))=0.0003932mol+0.0001057 mol/0.0027053 mol˜0.18

Molar ratio betweenn_(Na2HPO4.2H2O)+n_(NaH2PO4.2H2O/)n_(compound (ii))=0.0003932mol+0.0001057 mol/0.0027282 mol˜0.18

Molar ratio between n_(Na2HPO4.2H2O/)n_(compound (i))=0.0003932mol/0.0027053 mol˜0.145

Molar ratio between n_(Na2HPO4.2H2O/)n_(compound (ii))=0.0003932mol/0.0027282 mol˜0.144

Molar ratio between n_(NaH2PO4.2H2O/)n_(compound (i))=0.0001057mol/0.0027053 mol˜0.038

Molar ratio between n_(NaH2PO4.2H2O/)n_(compound (ii))=0.0001057mol/0.0027282 mol˜0.038

n_(compound (i))=m_(compound (i))/M_(compound (i))=0.710 g/240.27 gmol⁻¹=0.002955 mol

n_(compound (i))=m_(compound (i))/M_(compound (i))=0.590 g/240.27 gmol⁻¹=0.0024556 mol

n_(compound (i))=m_(compound (i))/M_(compound (i))=0.650 g/240.27 gmol⁻¹=0.0027053 mol

n_(compound (ii))=m_(compound (ii))/M_(compound (ii))=0.710 g/238.25 gmol⁻¹=0.00298 mol

n_(compound (ii))=m_(compound (ii))/M_(compound (ii))=0.590 g/238.25 gmol⁻¹=0.0024763 mol

n_(compound (ii))=m_(compound (ii))/M_(compound (ii))=0.650 g/238.25 gmol⁻¹=0.0027282 mol

n_(NACl)=m_(NACl)/M_(NACl)=0.380 g/58.44 g mol⁻¹=0.0065023 mol

n_(NACl)=m_(NACl)/M_(NACl)=0.320 g/58.44 g mol⁻¹=0.0054757 mol

n_(NACl)=m_(NACl)/M_(NACl)=0.350 g/58.44 g mol⁻¹=0.005989 mol

n_(Na2HPO4.2H2O)=m_(Na2HPO4.2H2O)/M_(Na2HPO4.2H2O)=0.077 g/177.99 gmol⁻¹=0.0004326 mol

n_(Na2HPO4.2H2O)=m_(Na2HPO4.2H2O)/M_(Na2HPO4.2H2O)=0.063 g/177.99 gmol⁻¹=0.0003539 mol

n_(Na2HPO4.2H2O)=m_(Na2HPO4.2H2O)/M_(Na2HPO4.2H2O)=0.07 g/177.99 gmol⁻¹=0.0003932 mol

n_(NaH2PO4.2H2O)=m_(NaH2PO4.2H2O)/M_(NaH2PO4.2H2O)=0.0185 g/156.01 gmol⁻¹=0.0001185 mol

n_(NaH2PO4.2H2O)=m_(NaH2PO4.2H2O)/M_(NaH2PO4.2H2O)=0.0145 g/156.01 gmol⁻¹=0.0000929 mol

n_(NaH2PO4.2H2O)=m_(NaH2PO4.2H2O)/M_(NaH2PO4.2H2O)=0.0165 g/156.01 gmol⁻¹=0.0001057 mol

n_(H2O)=m_(H2O)/M_(H2O)=0.17 g/18.02 g mol⁻¹=0.0094339 mol

n_(H2O)=m_(H2O)/M_(H2O)=0.11 g/18.02 g mol⁻¹=0.0061043 mol

n_(H2O)=m_(H2O)/M_(H2O)=0.14 g/18.02 g mol⁻¹=0.0077691 mol

In summary, a unit dosage as described herein may contain 650±60 mg ofthe free base of 4-Amino-7,8-dihydro-L-biopterin and/or of4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin, 140±30 mg of water ofcrystallization, 70±7 mg Na₂HPO₄.2H₂O, 16.5±2 mg NaH₂PO₄.2H₂O, and350±30 mg NaCl. It is also possible that a unit dosage of thecomposition contains 650±60 mg of the free base of4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin, 60±50 mg of water ofcrystallization, 70±7 mg Na₂HPO₄.2H₂O, 12±2.5 mg NaH₂PO₄.2H₂O, and350±30 mg sodium chloride (NaCl). In typical embodiments, this unitdosage refers to and is packaged for administration in a 50 ml vialsince after reconstitution with a volume of 50 ml pharmaceuticallyacceptable carrier such as water ad inject an infusion or injectionsolution that can be immediately administered is obtained. Thus, if forexample, a different unit dosage is used, for example a 30 ml vial, theamounts of all components of the composition are adjusted accordingly.In the example of a 30 ml vial, the amount of each component would bereduced to 3/5 (60%). That means, if a unit dosage in a 50 ml vialcontains 650±60 mg of the free base of 4-Amino-7,8-dihydro-L-biopterinand/or of 4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin, 140±30 mg ofwater of crystallization, 70±7 mg Na₂HPO₄.2H₂O, 16.5±2 mg NaH₂PO₄.2H₂O,and 350±30 mg NaCl, then a 30 ml unit dosage will contain 390±36 mg ofthe free base of 4-Amino-7,8-dihydro-L-biopterin and/or of4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin, 84±18 mg of water ofcrystallization, 42±4.2 mg Na₂HPO₄.2H₂O, 9.9±1.2 mg NaH₂PO₄.2H₂O, and210±18 mg NaCl. Accordingly, if a unit dosage in a 50 ml vial contains650±60 mg of the free base of 4-Amino-7,8-dihydro-L-biopterin and/or of4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin, 60±50 mg of water ofcrystallization, 70±7 mg Na₂HPO₄.2H₂O, 12±2.5 mg NaH₂PO₄.2H₂O, and350±30 mg NaCl, then a 30 ml unit dosage will contain 390±36 mg of thefree base of 4-Amino-7,8-dihydro-L-biopterin and/or of4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin, 36±18 mg of water ofcrystallization, 42±4.2 mg Na₂HPO₄.2H₂O, 7.2±1.2 mg NaH₂PO₄.2H₂O, and210±18 mg NaCl.

The pharmaceutical composition of the present invention can alsocomprise further molecules, for example, related compounds generatedduring the production process of the pharmaceutical composition of thepresent invention. Thus, the pharmaceutical composition of the presentinvention can comprise 1, 2, 3, 4, 5, 6, 7, or more additionalcompounds. These additional compounds may be selected from one or moreof the compounds including, but not limited to, of the group consistingof 4-Amino-7,8-dihydro-L-biopterin (which might be generated byspontaneous oxidation of 4-Amino-5,6,7,8-tetrahydro-L-biopterin, seeabove), 4-Amino-L-biopterin, (6R,S)-5,6,7,8-Tetrahydro-L-biopterin,1-[(6R,S)-2,4-Diamino-5,6,7,8-tetrahydropteridin-6-yl]propanol,1-[(6R,S)-2,4-Diamino-5,6,7,8-tetrahydropteridin-6-yl]propane,(1R,2S)-1-[(6R,S)-2-(Acetylamino)-4-amino-5,6,7,8-tetrahydropterin-6-yl]-1,2-diacetoxy-propane,2,4-Diamino-7,8-dihydropteridine, or 2,4-Diaminopteridine, to name a fewillustrative compounds.

As outlined above, pteridine derivatives have been shown to exhibitproperties different from that of other NO-inhibitors, making thecompound potentially more suitable than “classical” arginine analogues.Thus, the present invention also relates to a use of a lyophilizedpharmaceutical composition of the present invention for the treatment ofa disease. In some embodiments, the disease is selected from the groupconsisting of a traumatic brain injury, non-traumatic brain injury,preferably stroke or meningitis, elevated cranial pressure, secondarybrain injury.

The term “traumatic brain injury” or “brain trauma” occurs when anexternal force traumatically injures the brain. TBI can be classifiedbased on severity, mechanism (closed or penetrating head injury), orother features (e.g., occurring in a specific location or over awidespread area). A traumatic brain injury can occur as a consequence ofa focal impact upon the head, by a sudden acceleration/decelerationwithin the cranium or by a complex combination of both movement andsudden impact, as well as blast waves, or penetration by a projectile.The Glasgow Coma Scale (GCS), the most commonly used system forclassifying TBI severity, grades a person's level of consciousness on ascale of 3-15 based on verbal, motor, and eye-opening reactions tostimuli. In general, it is agreed that a TBI with a GCS of 13 or aboveis mild, 9-12 is moderate, and 8 or below is severe. Similar systemsexist for young children. From the diagnostic point of view, it isfurther distinguished between open and closed TBIs. An open TBI isconsidered to be an injury in which the cerebral meninges (Dura mater)is mechanically destroyed and the brain is in contact with theenvironment through this opening. Often, an open TBI is associated withthe exit of liquor and brain tissue debris. In a closed TBI the skull orcranium remains intact, and the primary damage of the brain (trauma) ischaracterized by local lesions such as contusions or hematomas and/ordiffuse cerebral tissue damage. The term “cranium” when referred toherein is the set of out of the neurocranium (braincase) and theviscerocranium (craniofacial) existing bony and cartilaginous headskeleton of vertebrates. “Intracranial” means within the cranium.

To the contrary, a “non-traumatic brain injury” does not involveexternal mechanical force to acquire a brain injury. Causes fornon-traumatic brain injury may include lack of oxygen, glucose, orblood. Infections can cause encephalitis (brain swelling), meningitis(meningeal swelling), or cell toxicity as e.g. caused by fulminanthepatic failure, as can tumours or poisons. These infections can occurthrough stroke, heart attack, near-drowning, strangulation or a diabeticcoma, poisoning or other chemical causes such as alcohol abuse or drugoverdose, infections or tumours and degenerative conditions such asAlzheimer's disease and Parkinson's disease. An acute neurodegenerativedisease is represented by “stroke”, which refers to the loss of brainfunction due to disturbances in the blood supply to the brain,especially when it occurs quickly, and is often associated withcerebrovascular disease. This can occur following ischemia (lack ofblood flow) caused by blockage (thrombosis, arterial embolism), or ahaemorrhage of central nervous system (CNS), or intracranialblood-vessels. As a result, the affected area of the brain cannotfunction normally.

Another disease which can be treated is “meningitis”, which is an acuteinflammation of the membranes covering the brain and spinal cord, knowncollectively as the meninges. The inflammation may be caused byinfection with viruses, bacteria, or other microorganisms, and lesscommonly by certain drugs.

In addition to the damage caused at the moment of injury, brain trauma(non-traumatic or traumatic brain injury) causes “secondary injury” orsecondary brain injury”, which refers to a variety of events that takeplace in the minutes and days following the injury. These processes,which include alterations in cerebral blood flow and the pressure withinthe skull, contribute substantially to the damage from the initialinjury. Secondary injury events may include for example damage to theblood-brain barrier, release of factors that cause inflammation, freeradical overload, excessive release of the neurotransmitter glutamate(excitotoxicity), influx of calcium and sodium ions into neurons, anddysfunction of mitochondria. Injured axons in the brain's white mattermay separate from their cell bodies as a result of secondary injury,potentially killing those neurons. Other factors in secondary injury arechanges in the blood flow to the brain; repeated transient disintegrityof the blood brain barrier; ischemia (insufficient blood flow); cerebralhypoxia (insufficient oxygen in the brain); cerebral oedema (swelling ofthe brain); and raised intracranial pressure (the pressure within theskull).

It can also be that the intracranial pressure may elevate due toswelling or a mass effect from a lesion, such as a haemorrhage. As aresult, cerebral perfusion pressure (the pressure of blood flow in thebrain) is reduced; ischemia results. When the pressure within the skullrises too high, it can cause brain death or herniation, in which partsof the brain are squeezed by structures in the skull. The term“intracranial pressure” (ICP) means the pressure inside the cranium andthus in the brain tissue and cerebrospinal fluid (CSF). The body hasvarious mechanisms by which it keeps the ICP stable, with CSF pressuresvarying by about 1 mmHg in normal adults through shifts in productionand absorption of CSF. ICP is measured in millimeters of mercury (mmHg)and, at rest, is normally 7-15 mmHg for a supine adult. Changes in ICPare attributed to volume changes in one or more of the compartmentscontained in the cranium. An “elevated pressure in the cranium” or“elevated intracranial pressure” means an increased pressure in thecranium of a subject in comparison to a normal, healthy subject. As theintracranial pressure is normally between 7-15 mm Hg; thus at 20-25 mmHg, the upper limit of normal, is already considered an elevatedintracranial pressure and a treatment to reduce this pressure may beneeded. Thus, as an elevated intracranial pressure can be considered anypressure higher that 20 mm Hg in the cranium of a supine subject,preferably a pressure is higher than 25 mm Hg, higher than 26 mm Hg,higher than 27 mm Hg, higher than 28 mm Hg, higher than 29 mm Hg, higherthan 30 mm Hg, higher than 31 mm Hg, higher than 32 mm Hg, higher than33 mm Hg, higher than 34 mm Hg or higher than 35 mm Hg.

Along this line, the present invention further relates to the use of thelyophilized pharmaceutical composition of the present invention in themanufacture of a medicament for treating a subject having closed headinjury, elevated cranial pressure and secondary brain injury. A “closedhead injury” when referred to herein means is a type of traumatic braininjury in which the cranium and dura mater remain intact (see alsoabove).

Also, the present invention relates to a method of treating a disease ina subject, comprising the step of administering a lyophilizedpharmaceutical composition of the present invention to a subject in needthereof.

For the administration, the attending physician and clinical factorswill determine the dosage regimen. As is well known in the medical arts,dosages for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. A typical daily dose canbe, for example, in the range of 2.5 mg/kg to 12.5 mg/kg body weight;however, doses below or above this exemplary range are also envisioned,especially considering the aforementioned factors.

The dosages are preferably given once a day for maximally 4 days,however, during progression of the treatment the dosages can be given inmuch longer time intervals and in need can be given in much shorter timeintervals, e.g., daily. In a preferred case the immune response ismonitored using herein described methods and further methods known tothose skilled in the art and dosages are optimized, e.g., in time,amount and/or composition. Dosages will vary but a preferred dosage forintravenous administration of compound (I) and/or compound (II) is fromapproximately 2.5 mg/kg to 12.5 mg/kg body weight and day.

If the regimen is an injection or a continuous short-term infusion, itshould also be in the range of about 1 μg to about 1 mg units perkilogram of body weight per minute, respectively. Progress can bemonitored by periodic assessment.

In vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of thedisease, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. Preferably, the pharmaceutical composition isadministered directly or in combination with an adjuvant.

The maximal daily dose (DDD) is based on the amount of the compound (I)and/or (II), which typically is used for the main indication of subjects(e.g. adults) per day. However, the dosage regimen may differ frominjections to infusions or even multiple injections and/or infusions aday. In some embodiments, in the method of treating a disease in asubject the maximal daily dose is 12.5 mg/kg body weight, preferably12.5, 11.5, 10.5, 9.5 8.5, 7.5, 6.5, 5.5, 4.5, 3.5, 2.5 mg/kg bodyweight. In some embodiments, the maximal daily dose is 10.0, 9.0 orpreferably 8.5 mg/kg body weight, or even lower such as 7.5, 5.0, 2.5mg/kg body weight.

As used herein, an interval which is defined as “(from) X to Y” equateswith an interval which is defined as “between X and Y”. Both intervalsspecifically include the upper limit and also the lower limit. Thismeans that for example an interval of “2.5 mg/kg to 12.5 mg/kg” or“between 2.5 mg/kg to 12.5 mg/kg” includes a concentration of 2.5, 3.5,4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5 and 12.5 as well as any givenintermediate value.

It is also envisaged that the pharmaceutical compositions are employedin co-therapy approaches, i.e. in co-administration with othermedicaments or drugs, for example other drugs for preventing, treatingor ameliorating diseases such as traumatic or non-traumatic braininjury.

“Subjects” that are treated in the present invention are preferablymammals such as humans, monkeys, cats, dogs, horses, pigs, cattle, miceor rat with humans being preferred.

The present invention also envisages a method for preparing alyophilized solid pharmaceutical composition (adapted for intravenousadministration) comprising

-   a) a compound having the formula (I):

-   -   and/or a compound having the formula (II):

-   and-   b) at least one phosphate salt and optionally NaCl;-   the method comprising:-   aa) dissolving the compound of the formula (III) and/or (II):

-   -   with a buffer, wherein preferably the buffer comprises the        phosphate; and

-   bb) lyophilization of the solution obtained in aa).

In this context, it is noted that while the compound of formula (II) isreadily available as such from synthesis, the typical synthesis productof the compound of formula (I) is its dihydrochloride dihydrate shown informula (III). For this reason, in the method of preparing a solidcomposition of the invention that contains the compound of formula (I),4-Amino-5,6,7,8-tetrahydro-L-biopterin, the starting material istypically 4-Amino-5,6,7,8-tetrahydro-L-biopterin dihydrochloridedihydrate, the compound of formula (III).

Also, the present invention further relates to a method for preparing alyophilized solid pharmaceutical composition (adapted for intravenousadministration) comprising

-   a) a compound having the formula (I):

-   -   and/or a compound having the formula (II):

-   and-   b) at least one inorganic acid, preferably NaCl;-   the method comprising:-   aa) dissolving the compound of the formula (III) and/or of the    formula (II):

-   -   with a buffer;

-   bb) lyophilisation of the solution obtained in aa).

The buffer as described in aa) is used to dissolve the compound of theformula (III) and/or of the formula (II) to give a compound-buffersolution. A buffer, in general, is a solution which contains a weak acid(or a base) and the salt of that weak acid (or that base). Further, itresists changes in pH when small quantities of an acid or an alkali areadded to it. Various phosphates may form several different buffercombinations. Three possible sodium phosphates may includeNaH₂PO₄—sodium dihydrogen phosphate, Na₂HPO₄—disodium hydrogenphosphate, Na₃PO₄—sodium phosphate.

The buffer in aa) can also be a sodium hydrogen phosphate buffercomprising at least one phosphate salt. A “sodium hydrogen phosphatebuffer” is a buffer comprising a sodium phosphate salt. In someembodiments, the sodium phosphate buffer comprises at least one sodiumphosphate salt as described above. In other embodiments, the sodiumphosphate buffer comprises 2, 3, 4, 5, or more sodium phosphate salts,preferably the two different phosphate salts as described above. Inanother embodiment of the method of the present invention, the sodiumhydrogen phosphate buffer is prepared by separately dissolvingNaH₂PO₄.2H₂O and Na₂HPO₄.2H₂O.

The term “separately dissolving” means that the dissolving of eachsodium phosphate is taking place spaced apart from each other, forexample in different glassware. In a further embodiment of the method ofthe present invention, the sodium hydrogen phosphate buffer has a pH of7.4 by adding the NaH₂PO₄.2H₂O dissolution to the Na₂HPO₄.2H₂O solution.

In addition the buffer in aa) can comprise NaOH, sodium hydrogenphosphate buffer and water. Optionally, the NaOH is a 5N NaOH solution.A “5N NaOH solution” means a solution of NaOH (sodium hydroxide) whichhas a normality of 5 (5N). If the concentration of a sodium hydroxidesolution is c (NaOH)=5 mol/L, then its normality is 5N.

In some embodiments of the method of the present invention the buffercomprises 12-16% (w/w) NaOH 5N, 8-12% (w/w) sodium hydrogen phosphatebuffer and 74-78% (w/w) water for injection.

In further embodiments of the method of the present invention, thebuffer has a pH of about 8, 9, 10, 11, 12, 13 or 14.

In yet another embodiment of the method of the present invention, thesolution in step aa) has a pH of about 4, 5, 6, 7, 8, 9, 10 or 11preferably between 6.5-7.6, most preferably about 7.4.

The buffer can be prepared in different ways. For example, it can beprepared under degasification to give a degassed buffer. A “degassedbuffer” refers to the removal of dissolved gases e.g. oxygen fromliquids, especially water or aqueous solutions. Bubbling a solution withan inert gas substitutes the dissolved harmful, reactive gases such asoxygen and carbon dioxide. Nitrogen, argon, helium, and other inertgases are commonly used. To complete the substitution, the solutionshould be stirred vigorously and bubbled for a long time. In someembodiments, the buffer is degassed with nitrogen until the oxygencontent is <1.0 ppm (parts per million).

As a further additional step in the method, the solution obtained in aa)can be sterile filtered, preferably with a 0.22 μm filter. Sterilefiltered embraces any process that eliminates (removes) or kills allforms of microbial life, including transmissible agents (such as fungi,bacteria, viruses, spore forms, etc.) present on a surface, contained ina fluid, in medication or in a composition (solid or fluid). Here,sterilization is achieved by filtration. Sterilization can also beachieved by applying heat, chemicals, irradiation, high pressure, orcombinations thereof with a filtration step. The filter can havedifferent pore sizes. Usually, a filter with pore size 0.2 μm(microfiltration) will effectively remove microorganisms. In theprocessing of Biologics, viruses must be removed or inactivated e.g. byheat or glutaraldehyde or the like. Nanofilters with a smaller pore sizeof 20-50 nm (nanofiltration) are used. The smaller the pore size thelower the flow rate. To achieve higher total throughput or to avoidpremature blockage, pre-filters might be used to protect small poremembrane filters.

In another embodiment of the method of the present invention after thepreparation of the pharmaceutical composition; the lyophilisation isstarted at most 2 hours later, preferably at most 1.5 hours later, 1hour later, 30 minutes later most preferably at most 15 minutes later.

Notably, the present invention also relates to a pharmaceuticalcomposition obtainable by the method of the present invention.

For a better handling of the solid pharmaceutical composition of thepresent invention the lyophilisate obtained in bb) or the pharmaceuticalcomposition of the present invention is filled into vials, preferably inan amount about 1-1.5 g, preferably about 1.25 g solid formulation.Alternatively, the lyophilisate obtained in bb) or the pharmaceuticalcomposition may be filled into vials in an amount about 0.9-1.4 g,preferably 1.15 g solid formulation. When used as “unit dosage, theamount of about 1-1.5 g, or preferably of about 1.25 g solidformulation, or, alternatively, the amount of about 0.9-1.4 g,preferably 1.15 g solid formulation is filled into a 50 ml vial. Theterm “vial” refers to a (small) glass or plastic vessel or bottle, oftenused to store pharmaceutical compositions as liquids or solids.Nowadays, vials are also often made of plastics such as polypropylene orpolystyrene. Any other suitable container can of course also be used forthe storage of the solid composition of the invention.

To obtain a ready-to use injectable solution, the method of the presentinvention also comprises the step of reconstituting the lyophilisateobtained in bb) in a pharmaceutically acceptable fluid for thepreparation of an injectable solution. The term “injectable solution”refers to a solution that can be utilized for intravenousadministration, preferably for injection or infusion (see also above thedisclosure for injection). An injectable solution comprises apharmaceutically acceptable fluid.

The present invention is further characterized by the following list ofitems:

Item 1. A solid pharmaceutical composition adapted for intravenousadministration comprising

-   a) a compound having the formula (I):

-   -   and/or a compound having the formula (II):

-   and-   b) at least one phosphate salt.

Item 2. The pharmaceutical composition of claim 1, wherein the at leastone phosphate salt is a sodium phosphate, a potassium phosphate or anammonium phosphate.

Item 3. The pharmaceutical composition of item 2, wherein the phosphatesalt is selected from the group consisting of Na₂HPO₄ (water free),Na₂HPO₄.2H₂O, Na₂HPO₄.7H₂O, Na₂HPO₄.12H₂O, NaH₂PO₄ (water free),NaH₂PO₄.H₂O, NaH₂PO₄.2H₂O, K₂HPO₄ (water free) K₂HPO₄.3H₂O, KH₂PO₄(water free) and mixtures thereof.

Item 4. The pharmaceutical composition of item 2, wherein the phosphatesalt is Na₂HPO₄.2H₂O and wherein the quantity of the Na₂HPO₄.2H₂Opresent in the composition is chosen such that the molar ratio of theNa₂HPO₄.2H₂O to compound (I) or compound (II) ranges from 0.04 to 0.4.

Item 5. The pharmaceutical composition of item 2, wherein the sodiumphosphate is NaH₂PO₄.2H₂O, and wherein the quantity of the NaH₂PO₄.2H₂Opresent in the composition is chosen such that the molar ratio of theNaH₂PO₄.2H₂O to compound (I) or compound (II) ranges from 0.01 to 0.09.

Item 6. The pharmaceutical composition of any of items 1-5, comprisingtwo different sodium phosphate salts.

Item 7. The pharmaceutical composition of item 6, wherein the twodifferent sodium phosphate salts are NaH₂PO₄.2H₂O and Na₂HPO₄.2H₂O.

Item 8. The pharmaceutical composition of item 7, wherein the quantityof the NaH₂PO₄.2H₂O and Na₂HPO₄.2H₂O present in the composition ischosen such that the molar ratio of both NaH₂PO₄.2H₂O and Na₂HPO₄.2H₂Oto compound (I) or compound (ii) ranges from 0.02 to 0.5.

Item 9. The pharmaceutical composition of any of items 1-8, wherein thecompound (I) and/or the compound (II) are present as the free base.

Item 10. The pharmaceutical composition of any of items 1-9, wherein thepharmaceutical composition is a lyophilized pharmaceutical composition.

Item 11. The pharmaceutical composition of any of items 1-10, whereinthe compound (I) is a compound having the formula (Ia):

Item 12. The pharmaceutical composition of any of items 1-11, whereinthe compound (II) is a compound having the formula (IIa):

Item 13. The pharmaceutical composition of any of items 1-12, whereinthe pharmaceutical composition comprises an additional pharmaceuticalexcipient.

Item 14. The pharmaceutical composition of item 13, wherein theadditional pharmaceutical excipient is an inorganic salt.

Item 15. The pharmaceutical composition of item 14, wherein theinorganic salt is selected from MgCl₂, CaCl₂, NH₄Cl, KCl, or NaCl.

Item 16. The pharmaceutical composition of item 15, wherein theinorganic salt is NaCl.

Item 17. The pharmaceutical composition of item 16, wherein the quantityof the NaCl present in the composition is chosen such that the molarratio of the NaCl to compound (I) or compound (II) ranges from 1.5 to 4,preferably from 1.8 to 3.7.

Item 18. The pharmaceutical composition of any of items 1-17, whereinthe composition further comprises crystallization water.

Item 19. The pharmaceutical composition of any of items 1-18, whereinthe composition is adapted to be reconstituted in water.

Item 20. The pharmaceutical composition of any of items 1-19, whereinthe composition is adapted for administration by infusion or injection.

Item 21. The pharmaceutical composition of any of items 1-20, whereinthe compound (I) is (6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin.

Item 22. The pharmaceutical composition of any of items 1-21, whereinthe compound (I) is (6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin.

Item 23. The pharmaceutical composition of any of items 1-22, whereinthe compound (I) is a diastereomeric mixture that comprises more(6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin than(6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin.

Item 24. The pharmaceutical composition of item 23, wherein the quantityof the (6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin and the(6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin is chosen such that theratio of the amount of (6R)-4-Amino-5,6,7,8-tetrahydro-L-biopterin tothe (6S)-4-Amino-5,6,7,8-tetrahydro-L-biopterin ranges from 0.5 to 2,preferably around 1.3.

Item 25. The pharmaceutical composition of any of items 1-24, wherein aunit dosage of the composition contains 650±60 mg of the free base of4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin, 140±30 mg of water ofcrystallization, 70±7 mg Na₂HPO₄.2H₂O, 16.5±2 mg NaH₂PO₄.2H₂O, and350±30 mg NaCl.

Item 26. The composition of any of items 1-25, wherein the compositioncomprises 1, 2, 3, 4, 5, 6, 7, or more additional compounds, wherein theadditional compounds are selected from the group consisting of one ormore of the compounds selected from the group consisting of4-Amino-L-biopterin, (6R,S)-5,6,7,8-Tetrahydro-L-biopterin,1-[(6R,S)-2,4-Diamino-5,6,7,8-tetrahydropteridin-6-yl]propanol,1-[(6R,S)-2,4-Diamino-5,6,7,8-tetrahydropteridin-6-yl]propane,(1R,2S)-1-[(6R,S)-2-(Acetylamino)-4-amino-5,6,7,8-tetrahydropterin-6-yl]-1,2-diacetoxy-propane,2,4-Diamino-7,8-dihydropteridine, 2,4-Diaminopteridine.

Item 27. Use of a lyophilized pharmaceutical composition of any of items1 to 26 for the treatment of a disease.

Item 28. The use of item 27, wherein the disease is selected from thegroup consisting of a traumatic brain injury, non-traumatic braininjury, preferably stoke or meningitis, elevated cranial pressure,secondary brain injury.

Item 29. A method for preparing a lyophilized solid pharmaceuticalcomposition adapted for intravenous administration comprising

-   a) a compound having the formula (I):

-   -   and/or a compound having the formula (II):

-   -   and    -   b) at least one phosphate salt; the method comprising:

-   aa) dissolving the compound of the formula (III) and/or (II):

-   -   with a buffer;

-   bb) lyophilization of the solution obtained in aa).

Item 30. The method of item 29, wherein the method further comprises thestep of reconstituting the lyophilisate obtained in bb) in apharmaceutically acceptable fluid for the preparation of an injectablesolution.

Item 31. The method of item 29 or 30, wherein the buffer in aa) is asodium hydrogen phosphate buffer comprising at least one phosphate salt.

Item 32. The method of any of items 29-31, wherein the buffer in aa)comprises NaOH, sodium hydrogen phosphate buffer and water.

Item 33. The method of item 31, wherein the NaOH is a 5N NaOH solution.

Item 34. The method of item 31 wherein the sodium hydrogen phosphatebuffer is prepared by separately dissolving NaH₂PO₄.2H₂O andNa₂HPO₄.2H₂O.

Item 35. The method of item 33 wherein the sodium hydrogen phosphatebuffer has a pH of 7.4 by adding the NaH₂PO₄.2H₂O solution to theNa₂HPO₄.2H₂O dissolution.

Item 36. The method of any of items 29-35, wherein the buffer comprises12-16% (w/w) NaOH 5N, 8-12% (w/w) sodium hydrogen phosphate buffer and74-78% (w/w) water for injection.

Item 37. The method of any of items 29-36, wherein the solution obtainedin aa) is sterile filtered, preferably with a 0.22 μm filter.

Item 38. The method of any of items 29-37, wherein the buffer has a pHof about 8, 9, 10, 11, 12, 13 or 14.

Item 39. The method of any of items 29-38, wherein the solution in stepaa) has a pH of about 4, 5, 6, 7, 8, 9, 10 or 11 preferably between6.5-7.6, most preferably 7.4.

Item 40. The method of any of items 29-39, wherein the lyophilisateobtained in bb) is filled into vials, preferably in an amount about1-1.5 g, preferably 1.25 g solid formulation.

Item 41. The method of any of items 29-40, wherein buffer is preparedwith degassed buffer.

Item 42.The method of any of items 29-41, wherein buffer is degassedwith nitrogen until the oxygen content is <1.0 ppm.

Item 43. The method of any of items 29-42, wherein after the preparationof the solution; the lyophilisation is started at most 2 hours later.

Item 44. Pharmaceutical composition obtainable by the method of any ofitems 29-43.

Item 45. Use of the lyophilized pharmaceutical composition of any ofitems 1-28 in the manufacture of a medicament for treating a subjecthaving closed head injury, elevated cranial pressure and secondary braininjury.

Item 46. A method of treating a disease in a subject, comprising thestep of administering a lyophilized pharmaceutical composition of any ofitems 1 to 28 to a subject in need thereof.

Item 47. The method of item 46, wherein the maximal daily dose is 20mg/kg body weight, preferably, 17.5, 15.0 or 12.5, 10, 8.5, 7.5, 5.0,2.5 mg/kg body weight.

EXAMPLES

The following examples further illustrate the invention. These examplesshould however not be construed as to limit the scope of this invention.The examples are included for purposes of illustration and the presentinvention is limited only by the claims.

Example 1—Description of Manufacturing Process

VAS203 is prepared in a multistep synthesis starting from commerciallyavailable L-biopterin. A process scheme is shown in FIG. 1. Thesynthesis of VAS203 is based on the publications W. Pfleiderer et al. inPteridines 1989, 1, 199-210 and Pteridines 1995, 6, 1-7.

It was the aim of the first development phase to reproduce theliterature procedures and to estimate their feasibility for multi-gramscale. Purification of intermediates by flash-chromatography waseliminated and substituted by precipitation or crystallisation steps.Furthermore, adoptions on molar ratios of reagents andreaction-conditions were performed, so that a robust process up to theintermediate MAE 119 (4-Amino-L-biopterin) was available.

It was problematic to establish a robust and reliable route for thehydrogenation of MAE 119 to VAS203, using PtO2 as catalyst. Differentsolvents and aqueous media at different pH-values were tested, but onlywhen diluted hydrochloric acid was used, quick and reproducible results,especially on the diastereomeric ratio of the drug substance, could beachieved. The total amount of residual platinum burden could bedrastically reduced by pre-hydrogenation of the platinum (IV) oxidecatalyst, thereby the diastereomeric ratio changed slightly. Workup ofthe acidic solution of VAS203 proved to be difficult, since simpleevaporation of water at reduced pressure left a glass-like residue. Thebreakthrough was achieved, when 2-propanol was used as co-solvent forthe evaporating process. This procedure yielded a solid, butunfortunately, 2-propanol is incorporated into this solid. Byconventional drying-conditions the amount of residual 2-propanol couldnot be reduced below 5% (w/w). To overcome this problem, a finallyophilisation step to generate VAS203 can be included. Single batchesmay be pooled to give larger quantities.

Evidence for the structural assignment of VAS203 was provided by theroute of synthesis, supported by elemental analysis, nuclear magneticresonance (¹H-NMR and ¹³C-NMR), UV and IR spectroscopy (data not shown).

Example 2 Description and Composition of the Drug Product

VAS203 will be supplied as a sterile, white to pale red or brownlyophilised powder filled in 50 mL glass vials under nitrogen as aprotective atmosphere. Each vial contains 650±60 mg of the free base of4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin and 140±30 mg of water ofcrystallisation. Additionally, the vials contain 350±30 mg sodiumchloride (NaCl), 70±7 mg disodium hydrogen phosphate dihydrate(Na₂HPO₄.2H₂O), and 16.5±2 mg sodium dihydrogen phosphate dihydrate(NaH₂PO₄.2H₂O). The limits of tolerance of the drug product compositionare relatively high (±10%). The reason for this is the variation of thehydrochloride content of VAS203. The hydrochloride content of VAS203varies from batch to batch up to 10% (from 2.03 HCl to 2.24 HCl). Duringthe preparation of the drug product the hydrochloride was neutralised inthe present invention by addition of sodium hydroxide andsodium-phosphate buffer to obtain an isotonic solution with aphysiological pH value. Therefore, also the content of the moleculesgenerated during neutralisation (sodium chloride, disodium hydrogenphosphate and sodium dihydrogen phosphate) varies according to thehydrochloride content of the respective VAS203 batch. The given limitsof tolerance are necessary to meet the specifications of the qualityrelevant parameters pH and osmolality. The qualitative composition of 1g VAS203 vials is listed in Table 2.

TABLE 2 Reference Component to standards Function4-Amino-(6R,S)-5,6,7,8- In house Active ingredienttetrahydro-L-biopterin Sodium chloride* Ph. Eur. Osmolarity Disodiumhydrogen phosphate Ph. Eur. Buffer dihydrate Sodium dihydrogen phosphatePh. Eur. Buffer dihydrate Water for injection (aqua ad Ph. Eur. Solventused for inject.) reconstitution Nitrogen Ph. Eur. Protective atmosphere*Sodium chloride is generated during the preparation of the formulationwhen the hydrochloride of the drug substance reacts with the sodiumhydroxide solution. Both ingredients comply with the EuropeanPharmacopoeia.

Pharmaceutical Development

A solid lyophilised dosage form is being developed here for VAS203 forthe preparation of an infusion solution.

1 g VAS 203 ad 10 g sodium hydroxide/sodium hydrogen phosphate solutionbuffer with a final pH of 7.4 was selected to be aseptically processed,sterilised by membrane filtration and filled into 50 mL glass vials.Subsequently, this solution was freeze-dried according to a selectedlyophilisation program that produced a lyophilised product withexcellent stability. In this solid composition, VAS 203 is present asfree base 4-Amino-5,6,7,8-tetrahydro-L-biopterin. The vials are closedunder nitrogen, sealed with freeze-drying stoppers and closed with whitevacuum closures. The excipients are added in order to provide anisotonic solution with physiological pH after reconstitution with 50 mLwater ad inject. The pH of the final isotonic solution is 6.5 to 7.6.The final concentration of the drug substance VAS203 is 20 mg/mL.

Description of Manufacturing Process and Process Controls

At a pH of 7.4 4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterindihydrochloride dihydrate (VAS203) dissolved in solution buffer is veryunstable and oxidises in its two metabolites4-Amino-7,8-dihydro-L-biopterin and 4-Amino-L-biopterin. Therefore, itis important that the VAS203 solution is prepared with degassed bufferand after preparation of the VAS203 solution the lyophilisation shouldbe started without delay.

To allow lyophilisation in 50 mL vials a concentrated VAS203 solutionbuffer has to be prepared (1 g VAS203 ad 10 g buffer). The solutionbuffer is prepared by mixing the stock solutions:

14% (w/w) solution of sodium hydroxide (NaOH) 5 N

10% (w/w) solution of sodium hydrogen phosphate buffer (NaPB)

500 mmol/L, pH 7.4

76% (w/w) water for injection

The sterile solution buffer is then degassed with nitrogen until theoxygen content is <1.0 ppm. For each vial 9.0 g of this solution bufferis used to dissolve 1.0 g VAS203. The solution buffer is filled into anitrogen-flushed flask and solid (lyophilized) VAS203 is carefully addedwithin 15 minutes under nitrogen as a protective atmosphere. After checkof pH-value the VAS203 solution is sterile filtered with a 0.22 μmMillipak 200 filter. The 0.22 μm filters are integrity tested after use.A sample of 100 mL is taken for testing the bioburden. The bioburdenlimit of the drug product solution before sterile filtration is definedas =10 cfu/100 mL. The remaining solution is filtered for a second timeand dispensed into 50 mL vials, 10 g per vial. The vials are sterilisedprior to filling by dry heat according to section 5.1.1. of the EuropeanPharmacopoeia. After the lyophilisation the vials are closed undernitrogen, sealed with freeze-drying stoppers and closed with whitevacuum closures. The VAS203 solution is prepared shortly beforefreeze-drying. The aseptic filling process has been validated usingmedia fill. A flow chart of the successive steps of the manufacturingprocess, indicating the components used for each step is shown in FIG.2. The result is a solid composition in which VAS203 is present as freebase 4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin.

Stability

A stability monitoring was carried out for 24 months. Samples werestored “long-term” at 5° C. (verum). An accelerated stability study wascarried out at 40° C./75% RH for 6 months (verum). The analyticalprocedures used in the stability program include tests for assay, purityand related substances. The specifications applied and the methods usedare the same as for drug product release.

A stability monitoring was carried out for at least 60 months. Table 3shows the conditions and the storage period of the stabilityinvestigation. Samples were stored “long-term” at 5° C. (verum). Anaccelerated stability study has been carried out at 40° C./75% RH for 6months (verum). Table 4 gives the test schedule for the different timepoints. The analytical procedures used in the stability programmeinclude tests for assay, purity and related substances. Thespecifications applied and the methods used are the same as for drugproduct release. Exemplary results of the stability study are shown inTable 5 (which is depicted as FIG. 4).

TABLE 3 Conditions of stability monitoring to be carried out StorageStorage Stability Time Points Condition Packaging Period (Months) 5° C.50 mL glass vials 60 Months Initial(Release), 3, 6, 9, 12, 18, 24, 36,48, 60 40° C./75% RH 50 mL glass vials  6 Months Initial(Release), 1, 3,6

TABLE 4 Test schedule at stability time points Tests to be MonthCondition carried out* Initial — A 1 accelerated (40° C.) B 3 long-term(5° C.) & accelerated (40° C.) B 6 long-term (5° C.) & accelerated (40°C.) A (accelerated), B (long-term) 9 long-term (5° C.) B 12 long-term(5° C.) A 18 long-term (5° C.) B 24 long-term (5° C.) B 36 long-term (5°C.) A 48 long-term (5° C.) A 60 long-term (5° C.) A *Tests to be carriedout are: A: Complete shelf-life specification B: Complete shelf-lifespecification without test items “sterility”, “endotoxins” and“particles”

Discussion

Stability data are available for the batch stored at 5° C. and at 40°C./75% RH, protected from light. Minor changes were observed for samplesstored at both temperatures for up to 24 months but these showed noconsistent trend and are believed to be within the range of analyticalvariability. No degradation products were formed. A slight increasecould be observed for the first metabolite (oxidation product) of thedrug substance (4-Amino-7,8-dihydro-L-biopterin). After 24 monthsstorage at 5° C. in 7 of 10 vials a total of 15 visible particles havebeen found.

Stability data are available for the batch stored at 5° C. and at 40°C./75% RH, protected from light. No significant changes were detectedfor samples stored at both temperatures for up to 36 month. Nodegradation products were formed.

Conclusion

VAS203 drug product vials are stable when stored at 5° C., protectedfrom light, for not less than 36 months. VAS203 drug product vials arestable when stored at 40° C./75% RH, protected from light, for not lessthan 6 months. Considering the above, a shelf life of 42 months is setfor 1 g of lyophilised VAS203 powder filled in 50 mL glass vials undernitrogen as a protective atmosphere. The storage instruction will be tostore the vials at 2-8° C., and the vials must be wrapped with aluminiumfoil and packed in cardboard boxes to protect them from light. Allreconstituted drug products should be inspected visually for particulatematter. The storage instruction will be to store the vials at 2-8° C.

The shelf-life may be extended if appropriate long-term and acceleratedstability data from the concurrent stability study for 1 g VAS203 vialsand 50 mL placebo vials are available and the results meet the currentspecifications. The shelf-life will be determined according to theprinciples described in the ICH Q1E guidance:

If no significant change is detected at accelerated conditions over 6months and long term data show little or no change over time and lowvariability, twice the period covered by real time data will be used asthe extended shelf life, but the shelf life may not exceed the length ofavailable long-term data by more than 12 months.

Example 3—Stability During Preparation for Administration and the Courseof the Infusion

A stability study at room temperature was carried out for the drugsubstance VAS203 to test for any degradation of the reconstitutedsolution in transparent 50 mL polypropylene (PP) syringes which are usedin clinical studies. During stability testing the PP syringes wereexposed to normal day-light. Solution content, purity, relatedsubstances and pH were monitored for 48 hours (initial, 6, 24, 48 hoursafter preparation). Table 6 displays the results of the stability test.

Discussion

The solution content (assay) remained constant within specified limits(650±60 mg) for 48 hours. During the tested period, the chromatographicpurity for both diastereomers was well within specified limits, nosignificant change could be observed. The relative content of relatedsubstances remained constant within specified limits for 48 hours. Nodegradation products were formed. A slight increase could be observedfor the first metabolite of VAS203 (4-Amino-7,8-dihydro-L-biopterin).However, this oxidation was possibly caused by residual oxygen in thesyringe, between 6 and 48 hours the change was insignificant. The pH ofreconstituted VAS203 solution remained constant within specified limitsfor 48 hours. No change could be observed. Light sensitivity was notobserved up to 48 hours.

Conclusion

Investigations carried out demonstrate that a reconstituted solution ofVAS203 is stable in its application device (50 mL PP syringe) at roomtemperature exposed to normal day-light for 48 hours. However, use-bydate was reduced to avoid microbiological contamination. Use-by date wasdefined as time point of preparation of the VAS203 solution plus 27hours.

TABLE 6 Stability of reconstituted solution of VAS203 in 50 mL PPsyringe (application device), stored at room temperature Specification(phase I Storage Period in hours Test Item clinical batch) 0 6 24 48Assay (HPLC)    650 ± 60 mg 615 mg 610 mg 621 mg 626 mg (calculated onanhydrous and chloride-free basis) Purity (HPLC) (6R)-4-Amino-5,6,7,8-57.0 ± 3.5 55.5 55.4 55.4 55.5 tetrahydro-L-biopterin [% area](6S)-4-Amino-5,6,7,8- 37.5 ± 3.5 39.0 38.5 38.4 38.1tetrahydro-L-biopterin [% area] Related substances (HPLC)*: A [% area]≤4.5 3.3 3.8 3.8 4.1 B [% area] ≤1.2 0.6 0.6 0.6 0.6 C [% area] ≤2.7 1.51.6 1.5 1.5 Further related substances, each ≤0.6 0.2 0.2 0.2 0.1 [%area] Further related substances, total ≤3.0 0.5 0.6 0.6 0.4 [% area]Reconstituted Solution^(§) pH 6.5 to 7.6 7.1 N.T. 7.1 7.1 N.T.: NotTested; ^(§)Reconstituted in 50 mL WFI *A:4-Amino-7,8-dihydro-L-biopterin; B: Σ(6R)-5,6,7,8-Tetrahydro-L-biopterin and(6S)-5,6,7,8-Tetrahydro-L-biopterin; C: Σ1-[(6R)-2,4-Diamino-5,6,7,8-tetrahydropteridin-6-yl]propanol and1-[(6S)-2,4-Diamino-5,6,7,8-tetrahydropteridin-6-yl]propanol

It must be noted that as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “a reagent” includes one ormore of such different reagents and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsdescribed herein.

All publications and patents cited in this disclosure are incorporatedby reference in their entirety. To the extent the material incorporatedby reference contradicts or is inconsistent with this specification, thespecification will supersede any such material.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or sometimes when used herein with the term “having”.

When used herein “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.In each instance herein any of the terms “comprising”, “consistingessentially of” and “consisting of” may be replaced with either of theother two terms.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

What is claimed is:
 1. A method of preparing4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride dihydrate,the method comprising hydrogenating 4-Amino-L-biopterin of Formula (A)

using PtO₂ in diluted HCl as catalyst.
 2. The method of claim 1, whereinhydrogenation is carried out in 0.5 M HCl.
 3. The method of claim 1,wherein pre-hydrogenated PtO2 is used as catalyst.
 4. The method ofclaim 2, wherein the evaporation of water from the hydrogenated productof Formula (A) is performed in the presence of a co-solvent.
 5. Themethod of claim 4, wherein the co-solvent is 2-propanol to yield a solidhaving the formula (B)


6. The method of claim 5, further comprising removing residual solventfrom the solid of formula (B) to yield4-Amino-(6R,S)-5,6,7,8-tetrahydro-L-biopterin dihydrochloride dihydrate.7. The method of claim 6, wherein removing of residual solvent iscarried out by lyophilisation.